The dystrophin glycoprotein complex links laminin in the extracellular matrix to the cell cytoskeleton. Loss of dystrophin causes Duchenne muscular dystrophy, the most common human X-chromosome-linked genetic disease. The α7β1 integrin is a second transmembrane laminin receptor expressed in skeletal muscle. Mutations in the α7 integrin gene cause congenital myopathy in humans and mice. The α7β1 integrin is increased in the skeletal muscle of Duchenne muscular dystrophy patients and mdx mice. This observation has led to the suggestion that dystrophin and α7β1 integrin have complementary functional and structural roles. To test this hypothesis, we generated mice lacking both dystrophin and α7 integrin (mdx/α7-/-). The mdx/α7-/- mice developed early-onset muscular dystrophy and died at 2-4 weeks of age. Muscle fibers from mdx/α7-/- mice exhibited extensive loss of membrane integrity, increased centrally located nuclei and inflammatory cell infiltrate, greater necrosis and increased muscle degeneration compared to mdx or α7-integrin null animals. In addition, loss of dystrophin and/or α7 integrin resulted in altered expression of laminin-α2 chain. These results point to complementary roles for dystrophin and α7β1 integrin in maintaining the functional integrity of skeletal muscle.

I. We investigate the importance of a refuge from fish predation to the abundance, species composition and seasonal succession of zooplankton. Thirty lakes representing a range of depths were sampled twice in summer for physical/chemical parameters and zooplankton community structure.2. We deHne the refuge from centrarchid predators to be that space between the thentKKline and the zone of anoxia. As lakes vary in rate of oxygen depletion from the hypt»limnion. the refuge size and lake depth are independent: refuge size decreases during the summer period.3. Lake depth and refuge size independently explain variation among lakes in zooplankton species composition, but seasonal community change within lakes is best predicted by loss of refuge size.4. Refuge size also explains the substantial variation in the relative dominance of the two major daphnid species. Lakes possessing a large refuge are dominated by D. ptUicaria: those with a small refuge are dominated by the smaller. D. lialeata mcndotac. We suggest that lakes of intermediate refuge size, which arc characterized by high species diversity, represent a more equitable balance of predation and competition.

The ␣7␤1 integrin is a laminin receptor that has been implicated in muscle disease and the development of neuromuscular and myotendinous junctions. Studies have shown the ␣7␤1 integrin is also expressed in nonskeletal muscle tissues. To identify the expression pattern of the ␣7 integrin in these tissues during embryonic development, ␣7 integrin chain knockout mice were generated by a LacZ knockin strategy. In these mice, expression from the ␣7 promoter is reported by ␤-galactosidase. From embryonic day (ED) 11.5 to ED14.5, ␤-galactosidase was detected in the developing central and peripheral nervous systems and vasculature. The loss of the ␣7 integrin gene resulted in partial embryonic lethality. Several ␣7 null embryos were identified with cerebrovascular hemorrhages and showed reduced vascular smooth muscle cells and cerebral vascularization. The ␣7 null mice that survived to birth exhibited vascular smooth muscle defects, including hyperplasia and hypertrophy. In addition, altered expression of ␣5 and ␣6B integrin chains was detected in the cerebral arteries of ␣7 null mice, which may contribute to the vascular phenotype. Our results demonstrate for the first time that the ␣7␤1 integrin is important for the recruitment or survival of cerebral vascular smooth muscle cells and that this integrin plays an important role in vascular development and integrity. Developmental Dynamics 234:11-21, 2005.

Cerebral hypoxia induces a profound angiogenic response in the central nervous system (CNS). Using a mouse model of chronic cerebral hypoxia, we previously demonstrated that angiogenic vessels in the hypoxic CNS show marked upregulation of the extracellular matrix (ECM) protein fibronectin, along with increased expression of its major receptor, α5β1integrin on brain endothelial cells (BEC). As cerebral hypoxia also leads to glial activation, the aim of the current study was to define the temporal relationship between BEC responses and glial cell activation in this model of cerebral hypoxia. This revealed that BEC fibronectin/α5β1 integrin expression and proliferation both reached maximal level after 4 days hypoxia. Interestingly, up to 4 days hypoxia, all dividing cells were BEC, but at later time-points proliferating astrocytes were also observed. GFAP staining revealed that hypoxia induced marked astrocyte activation that reached maximal level between 7–14 days hypoxia. As newly formed cerebral capillaries require ensheathment by astrocyte end-feet in order to acquire mature brain endothelium characteristics, we next examined how expression of astrocyte end-feet adhesion molecules is regulated by hypoxia. This showed that the astrocyte adhesion receptors α6β4 integrin and dystroglycan were both markedly upregulated, with a time-course that closely resembled astrocyte activation. Taken together, this evidence shows that cerebral hypoxia promotes first an endothelial response, in which fibronectin promotes BEC proliferation. This is then followed by an astrocyte response, involving astrocyte activation, proliferation and re-organization of astrocyte end-feet, which correlates with increased expression of astrocyte end-feet adhesion molecules.

Cerebral angiogenesis is an important adaptive response to hypoxia. As the alpha v beta 3 integrin is induced on angiogenic vessels in the ischemic central nervous system (CNS), and the suggested angiogenic role for this integrin in other systems, it is important to determine whether the alpha v beta 3 integrin is an important mediator of cerebral angiogenesis. alpha v beta 3 integrin expression was examined in a model of cerebral hypoxia, in which mice were subject to hypoxia (8% O(2)) for 0, 4, 7, or 14 days. Immunofluorescence and western blot analysis revealed that in the hypoxic CNS, alpha v beta 3 integrin was strongly induced on angiogenic brain endothelial cells (BEC), along with its ligand vitronectin. In the hypoxia model, beta 3 integrin-null mice showed no obvious defect in cerebral angiogenesis. However, early in the angiogenic process, BEC in these mice showed an increased mitotic index that correlated closely with increased alpha 5 integrin expression. In vitro experiments confirmed alpha 5 integrin upregulation on beta 3 integrin-null BEC, which also correlated with increased BEC proliferation on fibronectin. These studies confirm hypoxic induction of alpha v beta 3 integrin on angiogenic vessels, but suggest distinct roles for the BEC integrins alpha v beta 3 and alpha 5 beta 1 in cerebral angiogenesis, with alpha v beta 3 having a nonessential role, and alpha 5 beta 1 promoting BEC proliferation.

BackgroundStudies of cerebral ischemia and other neuroinflammatory states have demonstrated a strong association between new vessel formation and microglial recruitment and activation, raising the possibility that microglia may be involved in promoting angiogenesis. As endothelial cell proliferation is a fundamental early step in angiogenesis, the aim of this study was to test this hypothesis by examining the influence of microglial secreted factors on brain endothelial cell (BEC) proliferation using BrdU incorporation.MethodsPrimary cultures of mouse BEC, microglia and astrocytes were used in this study. Proliferation of BEC was examined by BrdU incorporation. ELISA was used to quantify TNF and TGF-β1 levels within cell culture supernatants.ResultsMicroglia regulated BEC proliferation in a biphasic manner; microglia conditioned medium (MG-CM) from resting microglia inhibited, while that from activated microglia promoted BEC proliferation. A screen of microglial cytokines revealed that BEC proliferation was inhibited by TGF-β1, but promoted by TNF. ELISA showed that TNF and TGF-β1 were both present in MG-CM, and that while TGF-β1 dominated in resting MG-CM, TNF levels were massively increased in activated MG-CM, shifting the balance in favor of TNF. Antibody-blocking studies revealed that the influence of MG-CM to inhibit or promote BEC proliferation was largely attributable to the cytokines TGF-β1 and TNF, respectively.ConclusionThis data suggests that microglial activation state might be an important determinant of cerebral angiogenesis; inhibiting BEC proliferation and neovascularization in the normal central nervous system (CNS), but stimulating the growth of new capillaries under neuroinflammatory conditions.

Abstract-VascularV ascular smooth muscle cell (VSMC) proliferation and migration are major underlying factors in the development and progression of various forms of cardiovascular disease, including atherosclerosis, postangioplasty restenosis, transplant arteriopathy, and pulmonary hypertension. 1 Vascular remodeling during disease or injury involves altered expression of extracellular matrix proteins and cell surface integrins. 2-4 After arterial injury, laminin expression is reduced and fibronectin accumulates around VSMCs. 5,6 These changes coincide with a phenotypic switch in which contractile VSMCs adopt a proliferative phenotype, possibly as part of a developmental program associated with wound repair. 2,4 Integrins are transmembrane mechanosensors that relay signals from the extracellular matrix to the cell cytoskeleton and/or cell signaling pathways to modulate cell shape, adhesion, differentiation, proliferation, and contraction. 7 Various integrins modulate cell proliferation, usually by crosstalk with proliferative cell signaling pathways or in cooperation with growth factor receptors. 8 The ␣7␤1 integrin is a major laminin-binding receptor in VSMCs, and expression of this integrin increases after differentiation. 9 Previous studies using blocking antibodies and peptides have demonstrated that the ␣7␤1 integrin mediates adhesion of VSMCs to laminin in vitro. 9,10 Expression of this integrin has also been shown to be modulated by chemically induced injury and platelet-derived growth factor in cultured rat VSMCs. 10,11 We have previously demonstrated that embryonic loss of the ␣7 integrin results in vascular defects and partial embryonic lethality, whereas in adult mice, loss of the ␣7 integrin results in VSMC hyperplasia. 12 Loss of the ␣7␤1 integrin in VSMCs leads to altered expression of other integrin chains, which may contribute to the vascular phenotype observed in ␣7 integrin-null mice. 12 These observations have led to the hypothesis that the ␣7␤1 integrin promotes the contractile phenotype of VSMCs, but the mechanism of this regulation is unclear.Activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase signaling Original

The ␣7␤1 integrin, dystrophin, and utrophin glycoprotein complexes are the major laminin receptors in skeletal muscle. Loss of dystrophin causes Duchenne muscular dystrophy, a lethal muscle wasting disease. Duchenne muscular dystrophy-affected muscle exhibits increased expression of ␣7␤1 integrin and utrophin, which suggests that these laminin binding complexes may act as surrogates in the absence of dystrophin. Indeed, mice that lack dystrophin and ␣7 integrin (mdx/␣7 ؊/؊ ), or dystrophin and utrophin (mdx/utr ؊/؊ ), exhibit severe muscle pathology and die prematurely. To explore the contribution of the ␣7␤1 integrin and utrophin to muscle integrity and function, we generated mice lacking both ␣7 integrin and utrophin. Surprisingly, mice that lack both ␣7 integrin and utrophin (␣7/utr ؊/؊ ) were viable and fertile. However, these mice had partial embryonic lethality and mild muscle pathology, similar to ␣7 integrin-deficient mice. Dystrophin levels were increased 1.4-fold in ␣7/utr ؊/؊ skeletal muscle and were enriched at neuromuscular junctions. Ultrastructural analysis revealed abnormal myotendinous junctions, and functional tests showed a ninefold reduction in endurance and 1.6-fold decrease in muscle strength in these mice. The ␣7/utr ؊/؊ mouse, therefore, demonstrates the critical roles of ␣7 integrin and utrophin in maintaining myotendinous junction structure and enabling force transmission during muscle contraction. Together, these results indicate that the ␣7␤1 integrin, dystrophin, and utrophin complexes act in a concerted manner to maintain the structural and functional integrity of skeletal muscle. (Am J Pathol